A Computational Study of Elongation Factor G (EFG) Duplicated Genes: Diverged Nature Underlying the Innovation on the Same Structural Template

BACKGROUND: Elongation factor G (EFG) is a core translational protein that catalyzes the elongation and recycling phases of translation. A more complex picture of EFG's evolution and function than previously accepted is emerging from analyzes of heterogeneous EFG family members. Whereas the gene duplication is postulated to be a prominent factor creating functional novelty, the striking divergence between EFG paralogs can be interpreted in terms of innovation in gene function. METHODOLOGY/PRINCIPAL FINDINGS: We present a computational study of the EFG protein family to cover the role of gene duplication in the evolution of protein function. Using phylogenetic methods, genome context conservation and insertion/deletion (indel) analysis we demonstrate that the EFG gene copies form four subfamilies: EFG I, spdEFG1, spdEFG2, and EFG II. These ancient gene families differ by their indispensability, degree of divergence and number of indels. We show the distribution of EFG subfamilies and describe evidences for lateral gene transfer and recent duplications. Extended studies of the EFG II subfamily concern its diverged nature. Remarkably, EFG II appears to be a widely distributed and a much-diversified subfamily whose subdivisions correlate with phylum or class borders. The EFG II subfamily specific characteristics are low conservation of the GTPase domain, domains II and III; absence of the trGTPase specific G2 consensus motif "RGITI"; and twelve conserved positions common to the whole subfamily. The EFG II specific functional changes could be related to changes in the properties of nucleotide binding and hydrolysis and strengthened ionic interactions between EFG II and the ribosome, particularly between parts of the decoding site and loop I of domain IV. CONCLUSIONS/SIGNIFICANCE: Our work, for the first time, comprehensively identifies and describes EFG subfamilies and improves our understanding of the function and evolution of EFG duplicated genes

Biochemical properties of the superoxide dismutase from the pathogenic bacterium Helicobacter pylori

Helicobacter pylori, a pathogenic aerotolerant bacterium colonizing the gastric mucosa, causes gastritis and ulcer and, if not eradicated, may lead to the development of gastric tumors. H. pylori resistance to moderate oxygen concentrations is mainly due to the key anti-oxidant role played by superoxide dismutase (HpSOD), the enzyme scavenging the toxic superoxide anions formed during oxygen consumption. The 3D structure of a recombinant form of HpSOD showed that this enzyme, belonging to the Fe-SOD family, contains an extended C–terminal tail, which is missing in other bacterial SODs and whose role remains obscure. Furthermore, in some cultures of H. pylori the endogenous HpSOD was found anchored to the flagellar sheath of the bacterium. In the present study the biochemical properties of the recombinant HpSOD were investigated to improve the knowledge on the enzyme functions, which could explain the H. pylori survival in the harsh conditions of the stomach. The research is also focused on the possible role played by the unusual C–terminal extension of HpSOD. The high specific activity (5000 U/mg) of the recombinant enzyme and its discrete heat resistance (T1/2 = 64C) ensure its functional efficiency. The effect of typical inhibitors and inactivators of SODs was investigated; while sodium azide caused only a low inhibition, hydrogen peroxide and peroxynitrite provoked a significant inactivation of HpSOD. It is known that the genes involved in glutathione biosynthesis are missing in the H. pylori genome; however, HpSOD undergoes a glutathionylation reaction by the oxidized form of glutathione. A mutagenic analysis aimed at the replacement of the two cysteine residues possessed by HpSOD allowed the identification of C79 as the target residue of the S–glutathionylation reaction. The production of a deleted form of HpSOD lacking the C–terminal extension of the enzyme is on the way, to check the effect on the biochemical properties of the enzyme

The thioredoxin system in the dental caries pathogen Streptococcus mutans and the food-industry bacterium Streptococcus thermophilus

The Streptococcus genus includes the pathogenic species Streptococcus mutans, the main responsible of dental caries, and the safe microorganism Streptococcus thermophilus, used for the manufacture of dairy products. These facultative anaerobes control the levels of reactive oxygen species (ROS) and indeed, both S. mutans and S. thermophilus possess a cambialistic superoxide dismutase, the key enzyme for a preventive action against ROS. To evaluate the properties of a crucial mechanism for repairing ROS damages, the molecular and functional characterization of the thioredoxin system in these streptococci was investigated. The putative genes encoding its protein components in S. mutans and S. thermophilus were analysed and the corresponding recombinant proteins were purified. A single thioredoxin reductase was obtained from either S. mutans (SmTrxB) or S. thermophilus (StTrxB1), whereas two thioredoxins were prepared from either S. mutans (SmTrxA and SmTrxH1) or S. thermophilus (StTrxA1 and StTrxA2). Both SmTrxB and StTrxB1 reduced the synthetic substrate DTNB in the presence of NADPH, whereas only SmTrxA and StTrxA1 accelerated the insulin reduction in the presence of DTT. To reconstitute an in vitro streptococcal thioredoxin system, the combined activity of the thioredoxin components was tested through the insulin precipitation in the absence of DTT. The assay functions with a combination of SmTrxB or StTrxB1 with either SmTrxA or StTrxA1. These results suggest that the streptococcal members of the thioredoxin system display a direct functional interaction between them and that these protein components are interchangeable within the Streptococcus genus. In conclusion, our data prove the existence of a functioning thioredoxin system even in these microaerophiles

Properties of the thioredoxin system in microaerophiles from the Streptococcus genus

The thioredoxin system, involved in the preservation of the reduced state of citoplasmic proteins, is composed of two enzymes, thioredoxin (TrxA) and thioredoxin reductase (TrxB). To investigate on the properties of this system in the Streptococcus genus, the strains S. mutans, a pathogen involved in the development of dental caries, and S. thermophilus, a non pathogenic species employed in food industry, were selected. These fermenting facultative anaerobes are aerotolerant, as they possess superoxide dismutase, the first key enzyme involved in the control of reactive oxygen species; on the other hand, S. mutans and S. thermophilus lack catalase. The redundant putative genes encoding TrxA and TrxB in the genome of S. mutans and S. thermophilus were studied. Their predicted amino acid sequence was analysed to exclude products without the typical CXXC consensun. One TrxB gene in each strain, whereas two and three genes for TrxA remained in S. mutans and S. thermophilus , respectively. The TrxB gene from these strains was cloned and expressed, together with one TrxA gene from each strain. The activity of the recombinant enzymes was tested. Each TrxB catalysed the NADPH-dependent reduction of dithiobis-nitrobenzoate, and each TrxA induced the insulin precipitation in the presence of dithiothreitol. When the combined activity of the homologous enzymes was tested in the presence of NADPH as electron donor and human insulin as the TrxA substrate, only the reconstitued S. thermophilus system was active, thus demonstrating the direct functional interaction between the two homologous components. For the reconstitution of the S. mutans system, the other putative TrxA genes from this strain will be analysed. The study of the molecular and biochemical properties of the recombinant enzymes is in progress

THE GLUTATHIONE BIOSYNTHESIS IN THE PSYCHROPHILE PSEUDOALTEROMONAS HALOPLANKTIS

Glutathione (GSH) plays a relevant role in the control of redox homeostasis even in philogenetically distant microbial species. The presence of GSH was recently hypothesized even in cold-adapted sources, on the basis of the effects produced by this thiol on some antioxidant enzymes from Pseudoalteromonas haloplanktis, a psychrophile isolated from the Antarctic sea. The possible existence of an enzyme system aimed at GSH biosynthesis in P. haloplanktis was investigated. This biochemical process involves the activity of the enzymes glutamyl-cysteine ligase (GshA) and glutathione synthetase (GshB). In the genome of P. haloplanktis two putative genes encoding GshA (PhGshAI and PhGshAII) and one encoding GshB (PhGshB) were identified. In order to characterize the first enzyme system for GSH biosynthesis in a psychrophilic source, recombinant forms of PhGshAII and PhGshB were obtained. It is known that each step leading to GSH from glutamate, cysteine and glycine involves the hydrolysis of one ATP molecule. Therefore, a convenient assay for determining the activity of each enzyme was set up, using the radiolabelled compound [γ32P]ATP. Concerning rPhGshB, the pH optimum of the activity was between 7.5 and 7.8. The affinity for ATP in the temperature range 10-30°C was evaluated. rPhGshB showed a significant activity even at low temperatures, whereas the Km ranges between 0.14 and 0.25 mM in the 10-30°C temperature range. The kcat values were analysed through the Arrhenius equation and the calculated energy of activation was 77 kJ/mol, a value unusually high for a psychrophilic enzyme. The heat inactivation profile of rPhGshB allowed the determination of a half-life of 10 min at 50.5°C, a value high for a psychrophilic enzyme, although not unusual for antioxidant enzymes. The energy of activation related to the inactivation process was 208 kJ/mol, as usually found for psychrophilic enzymes. The research is now focused on the characterization of rPhGshAII

Key amino acid positions involved in activity, heat stability and covalent modification of rat mitochondrial manganese superoxide dismutase

Mitochondrial Mn-SOD is encoded by the nuclear genome as a precursor headed by a signal peptide spanning 24 amino acid residues; this leader peptide is then removed for the entry of the mature enzyme in mitochondria. The rat mitochondrial Mn-SOD (ratSOD2), sharing 93% amino acid identity with the human counterpart, is an appropriate model to study the molecular and functional properties of this key mitochondrial enzyme. This investigation regards the role of some crucial amino acid positions of ratSOD2 regulating catalysis, reactivity and thermal resistance of the enzyme. In particular, the role of three amino acid residues, namely Q143, Y34 and S82, has been investigated. The study was carried out through the heterologous production of the mature form of ratSOD2 and its mutants obtained by site-directed mutagenesis on the corresponding gene. Six recombinant forms of the enzyme were produced, carrying the Q143 or H143 residue with or without the Y34F or S82A replacement. All proteins bound manganese and were organized as homotetramers. A 6-fold reduction of the activity was observed in ratSOD2 forms containing the H143 variant; on the other hand, the Y34F and S82A substitutions only caused a modest reduction of enzymatic activity, compared to the Q143 form. Heat inactivation studies showed the high thermo-tolerance of ratSOD2 and allowed an evaluation of the related activation parameters of inactivation. Compared to the Q143 variant, the H143 counterpart was significantly less heat stable and displayed moderately lower enthalpic and entropic factors; the Y34F substitution caused a moderate reduction of heat stability, whereas the S82A replacement slightly improved the thermo-tolerance of the Q143 variant; both substitutions significantly increased the enthalpic and entropic factors of heat inactivation, the greatest effect being observed with the S82A substitution. All recombinant forms of ratSOD2 were glutathionylated in Escherichia coli, a feature pointing to the high reactivity of ratSOD2 towards glutathione. Moreover, the S82 position of the enzyme was found phosporylated in an in vitro system containing human mitochondrial protein extracts as source of protein kinases. These data highlight the role played by some critical residues of ratSOD2 and suggest fine levels of regulation occurring in vivo

Properties of a Putative Cambialistic Superoxide Dismutase from the Aerotolerant Bacterium Streptococcus thermophilus Strain LMG 18311

The aerotolerance of the lactic – fermentative bacterium Streptococcus thermophilus is mainly based on the key antioxidant function of superoxide dismutase (StSOD). In this work, the comparison of recombinant StSOD (rStSOD) forms obtained from two different initiation triplets indicated that the enzyme from S. thermophilus strain LMG 18311 spans 201 residues. rStSOD is organised as a homodimer, even though protein aggregates are formed in concentrated solutions. The capability of binding and exchanging Fe or Mn in the active site classifies rStSOD as a putative cambialistic enzyme; the moderate preference for iron is counteracted by a 1.5-fold higher activity measured for the Mn-containing form. The enzyme is thermostable, being its half-inactivation time 10 min at 73.5°C; the energetic parameters of the heat inactivation process are regulated by the level of Mn cofactor. The effect of Mn content on the rStSOD sensitivity towards inhibitors and inactivators was also evaluated. Sodium azide acts as a weak inhibitor of rStSOD and its Mn content does not greatly affect this sensitivity. Concerning the physiological inactivator hydrogen peroxide, the Mn-enriched rStSOD displays a great resistance; a moderate sensitivity is instead observed in the presence of a low Mn content. Contrary to hydrogen peroxide, sodium peroxynitrite is a powerful inactivator, a behaviour enhanced in the Mn-enriched enzyme. All these results were compared with the corresponding data previously reported for the cambialistic SOD from the taxonomically related S. mutans. In S. thermophilus the regulation of the enzyme functions by the Mn content appears less relevant with respect to S. mutans

Regulation of the properties of superoxide dismutase from the dental pathogenic microorganism Streptococcus mutans by iron- and manganese-bound co-factor

Streptococcus mutans, the main pathogen involved in the development of dental caries, is an aerotolerant microorganism. The bacterium lacks cytochromes and catalase, but possesses other antioxidant enzymes, such as superoxide dismutase (SmSOD). Previous researches suggested that SmSOD belongs to the 'cambialistic' group, functioning with Fe or Mn in the active site. A recombinant SmSOD (rSmSOD) with a His-tail has been produced and characterised. Studies on metal uptake and exchange proved that rSmSOD binds either Fe or Mn as a metal co-factor, even though with a consistent preference for Fe accommodation. The analysis of several enzyme samples with different values of the Mn/Fe ratio in the active site proved that the type of metal is crucial for the regulation of the activity of rSmSOD. Indeed, differently from the significant preference for Fe displayed by the enzyme in the binding reaction, its Mn-form was 71-fold more active compared to the Fe-form. The rSmSOD was endowed with a significant thermostability, its half-inactivation occurring after 10 min exposure at 71 or 73 degrees C, depending on the bound metal. Moreover, the enthalpic and entropic contribution to the heat inactivation process of rSmSOD were strongly regulated by the Mn content of the enzyme. The effect of typical inhibitors/inactivators has been investigated. rSmSOD was inhibited by sodium azide, and its sensitivity increased in the presence of higher Mn levels. Concerning two physiological inactivators, the enzyme displayed a different behaviour, being quite resistant to hydrogen peroxide and significantly sensitive to sodium peroxynitrite. Furthermore, the Mn co-factor had an amplifying role in the regulation of this different sensitivity. These results confirm the cambialistic nature of SmSOD and prove that its properties are regulated by the different metal content. The adaptative response of S. mutans during its aerobic exposure in the oral cavity could involve a different metal uptake by SmSOD